positions by a 5-dimethylallyltryptophan synthase from
Aspergillus clavatus
Biochemical Characterization of Indole Prenyltransferases
FILLING THE LAST GAP OF PRENYLATION POSITIONS BY A 5-DIMETHYLALLYLTRYPTOPHAN SYNTHASE FROM ASPERGILLUS CLAVATUS
*□SReceived for publication, October 27, 2011, and in revised form, November 26, 2011 Published, JBC Papers in Press, November 28, 2011, DOI 10.1074/jbc.M111.317982
Xia Yu (于霞)‡1, Yan Liu (刘燕)‡§2, Xiulan Xie (谢秀兰)¶, Xiao-Dong Zheng (郑晓冬)§, and Shu-Ming Li (李书明)‡3 From the‡Institut fu¨r Pharmazeutische Biologie und Biotechnologie, Philipps-Universita¨t Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany, the§Department of Food Science and Nutrition, Zhejiang University, 310058 Hangzhou, Zhejiang, China, and the¶Fachbereich Chemie, Philipps-Universita¨t Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany
Background:Known indole prenyltransferases catalyzed regioselective prenylations at N-1, C-2, C-3, C-4, C-6, and C-7 of the indole ring.
Results:Recombinant 5-DMATS was assayed with tryptophan and derivatives in the presence of DMAPP.
Conclusion:5-DMATS prenylated indole derivatives at C-5.
Significance:5-DMATS fills the last prenylation gap of indole derivatives and could be used as a potential catalyst for chemoen-zymatic synthesis.
The putative prenyltransferase gene ACLA_031240 be-longing to the dimethylallyltryptophan synthase superfamily was identified in the genome sequence ofAspergillus clavatus and overexpressed inEscherichia coli. The soluble His-tagged protein EAW08391 was purified to near homogeneity and used for biochemical investigation with diverse aromatic sub-strates in the presence of different prenyl diphosphates. It has shown that in the presence of dimethylallyl diphosphate (DMAPP), the recombinant enzyme accepted very well sim-ple indole derivatives withL-tryptophan as the best substrate.
Product formation was also observed for tryptophan-con-taining cyclic dipeptides but with much lower conversion yields. In contrast, no product formation was detected in the reac-tion mixtures of L-tryptophan with geranyl or farnesyl diphos-phate. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific regular prenylation at C-5 of the indole nucleus of the simple indole deriv-atives. EAW08391 was therefore termed 5-dimethylallyltrypto-phan synthase, and it filled the last gap in the toolbox of indole prenyltransferases regarding their prenylation positions.Kmvalues of 5-dimethylallyltryptophan synthase were determined forL -tryp-tophan and DMAPP at 34 and 76M, respectively. Average turn-over number (kcat) at 1.1 sⴚ1was calculated from kinetic data of
L-tryptophan and DMAPP. Catalytic efficiencies of 5-dimethyl-allyltryptophan synthase forL-tryptophan at 25,588 sⴚ1䡠Mⴚ1and for other 11 simple indole derivatives up to 1538 sⴚ1䡠Mⴚ1provided
evidence for its potential usage as a catalyst for chemoenzymatic synthesis.
Prenylated indole alkaloids represent a group of natural prod-ucts with diverse chemical structures and are widely distributed in bacteria, fungi, plants, and marine organisms (1, 2). Because of their impressive pharmacological and biological activities as drugs or as toxins (1, 3), prenylated indole alkaloids attract the attention of scientists from different scientific disciplines, including chem-istry, ecology, biology, pharmacology, and biochemistry (1, 4 –7).
These compounds are hybrid molecules containing prenyl moi-eties derived from prenyl diphosphates and indole or indoline ring from tryptophan or its precursors (1, 5). Indole prenyltransferases catalyze the connection of these two characteristic structural fea-tures and contribute significantly to the structural diversity of the prenylated indole alkaloids. Significant progress has been achieved for molecular biological, biochemical, and structural biological investigations on different prenyltransferase groups, including indole prenyltransferases (1, 8, 9). By the end of October 2011, 20 indole prenyltransferases from bacteria and fungi have been char-acterized biochemically (1, 10 –15). These enzymes catalyzed the transfer of prenyl moieties onto nitrogen or carbon atoms at the indole ring resulting in formation of “regularly” or “reversely” pre-nylated derivatives (15). More interestingly, indole prenyl-transferases showed the usually broad substrate specificity but catalyzed regiospecific prenylation at different positions of indole or indoline rings (Fig. 1) (15). CymD from the marine actinobacteriumSalinispora arenicolacatalyzed the reverse prenylation at the indole nitrogen ofL-tryptophan (12), whereas FtmPT2 from the fungusAspergillus fumigatus catalyzed the regular prenylation of 12,13-dihydroxyfumit-remorgin C at this position (16). Regular and reverse C2-pre-nylations were observed for FtmPT1 (17) and FgaPT1 (18), both fromA. fumigatus, respectively. Prenylations at C-3 of cyclic dipeptides by AnaPT (19) and CdpC3PT (10), both from the fungusNeosartorya fischeri,lead to the formation of indoline derivatives with an␣- and-configured reverse
*This work was supported in part by Deutsche Forschungsgemeinschaft Grant Li844/4-1 (to S.-M. L.) and by the Deutscher Akademischer Austauschdienst within the Programme des Projektbezogenen Personenaustauschs.
The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) XM_001269816.
□S This article containssupplemental Tables S1–S4 and Figs. S1–S14.
1Recipient of a Ph.D. scholarship from the China Scholarship Council for fellowships.
2Recipient of a project-related person exchange program from the China Scholarship Council for fellowships.
3To whom correspondence should be addressed. Tel.: 49-6421-2822461; Fax:
49-6421-2825365; E-mail: shuming.li@staff.uni-marburg.de.
JANUARY 6, 2012 •VOLUME 287 • NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 1371
prenyl moiety, respectively. FgaPT2 and its orthologues from different fungi represent the first pathway-specific enzyme in the biosynthesis of ergot alkaloids and catalyzed the regular prenylation ofL-tryptophan at C-4 (3, 20). CpaD fromAspergillus oryzaecatalyzed regular C4-prenylation as well but used cyclo-acetoacetyl-L-tryptophan as substrate (21). IptA from a soil bacterium Streptomyces sp. was
reported to prenylate L-tryptophan at C-6 (13) and 7-DMATS4 from A. fumigatus at C-7 (22). An additional
4The abbreviations used are: 7-DMATS, 7-dimethylallyltryptophan synthase;
DMAPP, dimethylallyl diphosphate; 5-DMATS, 5-dimethylallyltryptophan synthase; HMBC, heteronuclear multiple-bond correlation spectroscopy;
FPP, farnesyl diphosphate; GPP, geranyl diphosphate.
FIGURE 1.Examples of indole prenyltransferases with different prenylation positions.
1372 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 2 •JANUARY 6, 2012
example of C7-prenyltransferase is CTrpPT fromA. oryzae, which used cyclo-L-Trp-L-Trp as the best substrate (11).
In summary, indole prenyltransferases with regioselectivity for N-1, C-2, C-3, C-4, C-6, and C-7 have already been identi-fied and characterized in detail (Fig. 1). However, a prenyltrans-ferase responsible for transferring a prenyl moiety to C-5 of the indole ring has not been reported prior to this study. However, database searching revealed the presence of a number of bio-logically active indole alkaloids carrying a prenyl moiety at C-5 in nature (Fig. 2). These compounds include simple prenylated indole derivatives like the antifungal compound 3,5-hexalobine D from the plantHexalobus crispiflorus(23) or the brominated tryptamine derivative flustramine D from the bryozoanFlustra foliacea(24). Semicochliodinol A from the fungus Chrysospo-rium merdaChrysospo-rium(25) and petromurin B from the fungus Petro-myces muricatus(26) are C5-prenylated indolyl benzoquinones derived from two tryptophan molecules (27). Semicochliodinol A was reported to inhibit HIV-1 protease (25). C5-prenylated derivatives were also found for tryptophan-containing cyclic dipeptides like cyclo-L-Trp-L-Ala,e.g.echinulin from Aspergil-lus strains (28) or tardioxopiperazine A from the fungus Microascus tardifaciens(29). A relatively large indole alkaloid group are the indole diterpenes fromPenicillium(30) and other Ascomycetes (31). The members of this group carry prenyl moieties at C-5 (21-isopentenylpaxilline) (32), C-6, or at both positions (shearinine K) (30), or modified structures thereof.
The discrepancy between the natural occurrence of a large number of C5-prenylated indole derivatives on the one hand and undiscovered C5-prenyltransferases on the other hand prompted us to search for such enzymes. In this study, we reported the identification and characterization of the first C5-prenyltransferase of indoles,i.e.5-dimethylallyltryptophan synthase (5-DMATS) fromAspergillus clavatusand its poten-tial usage as a catalyst for chemoenzymatic synthesis.
EXPERIMENTAL PROCEDURES
Computer-assisted Sequence Analysis—Sequence identities were obtained by alignments of amino acid sequences using the program “BLAST 2 SEQUENCES” (www.ncbi.nlm.nih.gov).
FGENESH from Softberry and the DNASIS software package
(version 2.1, Hitachi Software Engineering, San Bruno, CA) were used for exon prediction and sequence analysis, respectively.
Chemicals—Dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), and farnesyl diphosphate (FPP) were pre-pared according to the method described for geranyl diphos-phate by Woodsideet al.(33). Indole derivatives of the highest available purity were purchased from TCI, Acros Organics, Aldrich, Sigma, Bachem, and Alfa Aesar.
Bacterial Strains, Plasmids, and Culture Conditions—
pGEM-T Easy and pQE70 were obtained from Promega and Qia-gen (Hilden, Germany), respectively.Escherichia coliXL1 Blue MRF⬘ (Stratagene, Amsterdam, the Netherlands) and M15 [pREP4] (Qiagen) were used for cloning and expression experi-ments, respectively. They were grown in liquid Terrific-Broth (TB) or Luria-Bertani (LB) medium and on solid LB medium with 1.5%
(w/v) agar at 37 or 22 °C. 50g䡠ml⫺1of carbenicillin were used for selection of recombinantE. coliXL1 Blue MRF⬘strains. Addition of carbenicillin at 50g䡠ml⫺1and kanamycin at 25g䡠ml⫺1was used for selection of recombinantE. coliM15 [pREP4] strains.
Cultivation of A. clavatus, RNA Isolation, and cDNA Synthesis—A. clavatusNRRL1 was kindly provided by Agricul-tural Research Service Culture Collection, United States Department of Agriculture, and was cultivated on solid YME media consisting of 0.4% (w/v) yeast extract, 1% (w/v) malt extract, 0.4% (w/v) glucose, pH 7.3, and 2% (w/v) agar at 26 °C.
For RNA isolation, mycelia ofA. clavatusNRRL1 from plates were inoculated into a 300-ml Erlenmeyer flask containing 100 ml of liquid YME media and cultivated at 26 °C and 160 rpm for 7 days. After separation of mycelia from the medium, RNA was isolated by using the High Pure RNA isolation kit (Roche Diag-nostics) according to the manufacturer’s protocol. cDNA was synthesized with the Transcriptor High Fidelity cDNA synthe-sis kit (Roche Diagnostics).
PCR Amplification and Gene Cloning—PCR amplification was carried out on a MiniCycler from Bio-Rad. A PCR fragment of 1300 bp containing the entire coding sequence of5-dmats was amplified from cDNA by using Expand high fidelity kit (Roche Diagnostics). The primers were 5-DMATS_for (5⬘ -FIGURE 2.Examples of naturally occurring C5-prenylated indole alkaloids.
JANUARY 6, 2012 •VOLUME 287 • NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 1373
GCCCAGC/ATGCCTCACCAAAACAGC-3⬘) at the 5⬘-end, and 5-DMATS_rev (5⬘- GGTCGAAGATCTCAATTTCCAA-GACTT-3⬘) at the 3⬘-end of the gene. Bold letters represent mutations inserted in comparison with the original genome sequence to give the underlined restriction site SphI at the start codon in 5-DMATS_for and BglII located at the predicted stop codon in 5-DMATS_rev. A program of 30 cycles with annealing at 58 °C for 50 s and elongation at 72 °C for 90 s was used for PCR amplification. The PCR fragment was cloned into pGEM-T easy vector resulting in plasmid pYL08, which was subsequently sequenced (Eurofins MWG Operon, Ebersberg, Germany) to confirm the sequence. Plasmid pYL08 was digested with BglII alone or together with SphI to obtain BglII-BglII fragment of 748 bp and SphI-BglII-BglII fragment of 531 bp, respectively. To get the expression construct pYL09, these two fragments were cloned into pQE70 subsequently.
Overproduction and Purification of His6-5-DMATS—For overproduction of 5-DMATS,E. coliM15 [pREP4] cells har-boring the plasmid pYL09 were cultivated in 2000-ml Erlen-meyer flasks containing 1000 ml of liquid TB medium, supple-mented with carbenicillin (50 g䡠ml⫺1) and kanamycin (25
g䡠ml⫺1), and then grown at 37 °C to an absorption at 600 nm of 0.6. For induction, isopropyl thiogalactoside was added to a final concentration of 0.4 mM, and the cells were cultivated for a further 16 h at 22 °C before harvest. The bacterial cultures were centrifuged, and the pellets were resuspended in lysis buffer (10 mMimidazole, 50 mMNaH2PO4, 300 mMNaCl, pH 8.0) at 2–5 ml/g wet weight. After addition of 1 mg䡠ml⫺1lysozyme and incubation on ice for 30 min, the cells were sonicated six times for 10 s each at 200 watts. To separate the cellular debris from the soluble proteins, the lysate was centrifuged at 13,000⫻gfor 30 min at 4 °C. One-step purification of the recombinant His6 -tagged fusion protein by affinity chromatography with nickel-nitrilotriacetic acid-agarose resin (Qiagen) was carried out according to the manufacturer’s instructions. The protein was eluted with 250 mMimidazole in 50 mMNaH2PO4, 300 mM
NaCl, pH 8.0. To change the buffer, the protein fraction was passed through a PD-10 column (GE Healthcare), which had been equilibrated with 50 mMTris-HCl, 15% (v/v) of glycerol, pH 7.5, previously. 5-DMATS was eluted with the same buffer and frozen at⫺80 °C for enzyme assays.
Protein Analysis and Determination of Relative Molecular Mass of Active His6-5-DMATS—Proteins were analyzed by 12%
(w/v) SDS-polyacrylamide gels according to the method of Laemmli (34) and stained with Coomassie Brilliant Blue G-250.
TheMr value of the recombinant active His6-5-DMATS was determined by size exclusion chromatography on a HiLoad 16/60 Superdex 200 column (GE Healthcare), which had been equilibrated with 50 mMTris-HCl buffer, pH 7.5, containing 150 mMNaCl. The column was calibrated with dextran blue 2000 (2000 kDa), ferritin (440 kDa), aldolase (158 kDa), conal-bumin (75 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa) (GE Healthcare). The proteins were eluted with the same buffer as for equilibration.
Enzyme Assays with 5-DMATS—The enzyme reaction mix-tures for determination of the relative activities with different indole derivatives orL-tyrosine (100l) contained 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 1 mM aromatic substrate, 2 mM
DMAPP, GPP, or FPP, 0.15–5% (v/v) glycerol, 0 –5% (v/v) DMSO, and 1Mpurified recombinant protein. The reaction mixtures were incubated at 37 °C for 7 h (with DMAPP) or 24 h (with GPP or FPP). The enzyme reactions were terminated by addition of 100l of methanol per 100l reaction mixture.
For determination of kinetic parameters of DMAPP,L -tryp-tophan at 1 mMand DMAPP at final concentrations of up to 3 mM were used as substrates. For determination of kinetic parameters ofL-tryptophan and derivatives, the assays con-tained 2 mMDMAPP. Because of the difference of solubility in the aqueous system, various concentrations were used for aro-matic substrates as follows: for8a and12a up to 5 mM, for 1a–3a,7a,and9a–11aup to 2 mM, and for4a– 6aup to 1 mM. The protein concentration was 20 nM(1a), 40 nM(DMAPP), or 200 nM(other substrates), and the incubation time was 30 min (1a), 40 min (DMAPP) or 60 min (other substrates).
Preparative Synthesis of Enzyme Products for Structure Elucidation—For isolation of the enzyme products, reactions were carried out in large scale (10 ml) containing each of the 12 substrates1a–12a(1 mM), DMAPP (2 mM), CaCl2(5 mM), Tris-HCl (50 mM, pH 7.5), glycerol 0.15–5% (v/v), and 5-DMATS (1.4M). After incubation for 16 h, the reactions were termi-nated by addition of 10 ml of methanol each. After removal of the precipitated protein by centrifugation at 6000 rpm for 30 min, the reaction mixtures were concentrated on a rotating vacuum evaporator at 30 °C to a final volume of 1 ml before injection onto HPLC.
HPLC Conditions for Analysis and Isolation of Enzyme Products—The enzyme products of the incubation mixtures were analyzed by HPLC on an Agilent series 1200 by using a Multiphor 120 RP-18 column (250⫻4 mm, 5m, C⫹S Chro-matographie Service, Langerwehe, Germany) at a flow rate of 1 ml䡠min⫺1. Water (solvent A) and methanol (solvent B) each with 0.5% (v/v) trifluoroacetic acid were used as solvents. For analysis of enzyme products of tryptophan, simple indole deriv-atives andL-tyrosine, a linear gradient of 20 –100% (v/v) solvent B, for 15 min were used. The column was then washed with 100% (v/v) solvent B for 5 min and equilibrated with 20% (v/v) solvent B for 5 min. For analysis of enzyme products of trypto-phan-containing cyclic dipeptides, the gradient began with 40%
(v/v) solvent B and increased from 40 to 100% (v/v) solvent B in 15 min. After washing with 100% (v/v) solvent B for 5 min, the column was equilibrated with 40% (v/v) solvent B after each run. For assays with GPP or FPP, a linear gradient of 20 – 84%
(v/v) solvent B for 12 min and then 84 –100% (v/v) solvent B for 18 min was used. The column was then washed with 100% (v/v) solvent B for 10 min and equilibrated with 20% (v/v) solvent B for 5 min. Detection was carried out with a Photo Diode Array Detector.
For isolation of the enzyme products, the same HPLC equip-ment with a Multospher 120 RP-18 column (250⫻10 mm, 5
m, C⫹S Chromatographie Service) was used. The flow rate was 2.5 ml䡠min⫺1. Water (solvent C) and methanol (solvent D) without acid were used as solvents. Gradients of 20 –100% (v/v) solvent D for different times were used for isolation. The col-umn was then washed with 100% (v/v) solvent D for 8 min and equilibrated with 20% (v/v) solvent D for 8 min.
1374 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 2 •JANUARY 6, 2012
NMR Spectroscopic Analysis and High Resolution ESI-MS—
1H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer or a JEOL ECX-400 spectrometer. The HSQC and HMBC spectra were recorded with standard methods (35) on the Bruker Avance 500 MHz spectrometer. Chemical shifts were referenced to the signal of CD3OD at 3.31 ppm or DMSO-d6 at 2.50 ppm. All spectra were processed with MestReNova 5.2.2.
The isolated products were also analyzed by high resolution ESI-MS with a Q-Trap Quantum (Applied Biosystems). Posi-tive ESI-MS data are given insupplemental Table S3.
Nucleotide Sequence Accession Number—The coding se-quence of 5-dmats(ACLA_031240) is available at GenBankTM under the accession number XM_001269816.
RESULTS
Sequence Analysis and Cloning of 5-Dimethylallyltryptophan Synthase Gene 5-dmats—In the course of our search for prenyl-transferases responsible for prenylation of tryptophan or indole derivatives at C-5, one putative geneACLA_031240from the genome sequence ofA. clavatusNRRL1 raised our interest. The deduced gene product EAW08391 consists of 427 amino acids and shares high sequence similarities with C4-prenyltrans-ferases of tryptophan, e.g.52% identity with FgaPT2 fromA.
fumigatus(20) at the amino acid level. Based on this homology, it can be expected that EAW08391 catalyzes a similar reaction as FgaPT2.
Inspection of down- and upstream genes ofACLA_031240in the genome sequence ofA. clavatus(www.ncbi.nlm.nih.gov) revealed that the enzymes encoded by these genes are very likely involved in the fungal development and biosynthesis of primary rather than secondary metabolites, e.g. prenylated indole alkaloids. This means thatACLA_031240is very likely not clustered with genes for the biosynthesis of secondary metabolites. For example, in the downstream sequence of ACLA_031240, the putative geneACLA_031250is separated from ACLA_031240 by a segment of 13 kb and encodes a putative MYB family conidiophore development protein.
ACLA_031260, downstream ofACLA_031250,was predicted to be a glycosyl hydrolase gene. In the upstream sequence of ACLA_031240, ACLA_031230was predicted to encode a UDP-N-acetylglucosaminyltransferase involved in the cell wall bio-synthesis.ACLA_031220 is likely a 60 S ribosomal protein gene.
Blast search with EAW08391 from A. clavatus in Gen-BankTMindicated the presence of three homologues as follows:
XP_001818057 encoded by AOR_1_1880174 fromA. oryzae RIB40; EED57628 encoded byAFLA_083250fromAspergillus flavusNRRL3357, and EEQ32624 encoded byMCYG_05443 fromArthroderma otaeCBS113480. EAW08391 shares on the amino acid level sequence identities of 76, 75, and 74% with XP_001818057, EED57628, and EEQ32624, respectively. It can be expected that these three enzymes catalyze the same reac-tion as EAW08391.
Inspection of down- and upstream genes ofAOR_1_1880174 in the genome sequence ofA. oryzae,AFLA_083250ofA. flavus, and MCYG_05443 of A. otae (www.ncbi.nlm.nih.gov) indi-cated also their nonclustering with other secondary metab-olite biosynthesis genes. The direct neighboring genes of
AOR_1_1880174were predicted to encodeL-arabitol/xylitol dehydrogenase (AOR_1_1878174) and a putative trans-porter gene for choline,␥-aminobutyric acid, or other amino acids (AOR_1_1882174). Orthologues of these two genes (AFLA_083240andAFLA_083270) were also found directly atAFLA_083250. A gene AFLA_083260coding for a small hypothetical protein with 84 amino acids was located betweenAFLA_083250andAFLA_083270. In the genome of A. otae, MCYG_05443was found between anL-allothreonine aldolase gene (MCYG_05442) and a hypercellular protein HypA gene (MCYG_05444).
To prove the function of EAW08391, the coding region of ACLA_031240, termed5-dmatsin this study, was amplified by PCR from cDNA synthesized from mRNA. The PCR product was cloned via pGEM-T easy vector into pQE70, resulting in the expression construct pYL09. For overproduction of His6 -5-DMATS,E. coliM15 cells harboring pYL09 were cultivated in TB medium and induced with 0.4 mMisopropyl thiogalactoside at 22 °C for 16 h. A significant band with migration near the 45-kDa size marker was observed on SDS-PAGE of the purified protein (seesupplemental Fig. S1), corresponding to the calcu-latedMrvalue of 50,411 for His6-5-DMATS. The yield was cal-culated to be 1.8 mg of purified protein/liter of culture. TheMr value of the native recombinant His6-5-DMATS was deter-mined by size exclusion chromatography at about 79,000. This indicated that 5-DMATS acted likely as a homodimer.
5-DMATS Accepted WellL-Tryptophan and Simple Indole Derivatives as Substrates—Because of the high sequence simi-larity with tryptophan C4-prenyltransferases mentioned above,
L-tryptophan and 17 simple indole derivatives (Table 1 and sup-plemental Table S1) were incubated with the purified 5-DMATS in the presence of DMAPP (2 mM). These sub-stances included eight tryptophan derivatives with modifica-tion at the indole ring and nine at the side chain. The reacmodifica-tion mixtures were incubated with 5-DMATS at a final concentra-tion of 1Mfor 7 h. HPLC analysis was used for monitoring the enzyme product formation. Assays with heat-inactivated pro-tein by boiling for 20 min were used as negative control.
HPLC analysis of the incubation mixtures showed clear product formation for 17 of the 18 tested indole derivatives with L-tryptophan as the best substrate (Table 1 and supple-mental Table S1). HPLCs of incubation mixtures of 12 sub-strates (1a–12a, Table 1) showed clearly the presence of one product peak for each substrate. Similar behavior was also observed for other five accepted substrates (data not shown).
Under this condition,L-tryptophan was almost completely con-sumed, and the 11 other substrates (2a–12a) were accepted with total yields between 38 and 91%. Even in the incubation mixtures with 0.40Mprotein for 1 h, the conversion yield for
L-tryptophan was calculated to be 95.4% (see supplemental Table S2).
Inspection of the activities of the tested substances (Table 1 andsupplemental Table S1) revealed that, with the exceptions for C5-substituted derivatives, all of the substances with mod-ifications by fluoro or methyl group at the indole ring were well accepted by 5-DMATS. These results could indicate the prenyl-ation position at C-5 of the indole rings of the accepted substrates. 5-Bromo-DL-tryptophan was not accepted by
JANUARY 6, 2012 •VOLUME 287 • NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 1375
5-DMATS. The conversion yields of 5-fluoro-L-tryptophan and 5-methyl-DL-tryptophan at 7.4 and 1.7%, respectively, were sig-nificantly lower than those with substitution at other positions of the indole ring. The prenylation of these substances had very likely taken place at other positions rather than C-5 (see under
“Discussion”). Modification at the side chain of tryptophan reduced enzyme activity, especially by shortening the chain length, as in the case of indole-3-acetic acid and tryptamine. In comparison, changes at the amino group such as N-methyla-tion (L-abrine,7a), replacement by a hydroxyl group (DL -indole-3-lactic acid, 10a), or deamination (indole-3-propionic acid, 11a) showed less influence on the enzyme activity.
A previous study (36) showed that the tryptophan C4-prenyl-transferase FgaPT2 also accepted tryptophan-containing cyclic dipeptides as substrates. 5-DMATS was therefore also assayed with five such cyclic dipeptides and analyzed on HPLC. It has been shown that these compounds were also substrates for
5-DMATS but were accepted with significantly lower yields (⬍10%) than most of the simple indole derivatives (see supple-mental Table S1). Incubations of 5-DMATS withL-tyrosine in the presence of DMAPP or withL-tryptophan in the presence of GPP or FPP did not result in the formation of any enzyme prod-uct, even after incubation with 1Mprotein for 24 h (see sup-plemental Table S2).
5-DMATS Catalyzed the Regular C5 Prenylation at the Indole Ring—To confirm the prenylation position, enzyme products of 12 substrates (1a–12a, Table 1) were isolated on HPLC and subjected to high resolution MS and NMR analyses.
High resolution ESI-MS (see supplemental Table S3) con-firmed the presence of one dimethylallyl moiety each in the products of1a–12aby detection of masses, which are 68 dal-tons larger than those of the respective substrates.
In the1H NMR spectra of all the enzyme products (taken in CD3OD or DMSO-d6), signals at␦H3.34 –3.47 (d, 2H-1⬘), 5.17–
5.40 (t or m, H-2⬘), 1.71–1.78 (s, 3H-4⬘), and 1.67–1.77 (s, 3H-5⬘) were observed (seesupplemental Table S4and supple-mental Figs. S3–S14), proving unequivocally the attachment of a regular dimethylallyl moiety to a carbon atom (37, 38).
Substrates 2a and7a–11a are derivatives ofL-tryptophan (1a) with modifications at N-1 at the indole ring or at the side chain. Characteristic signals of the four coupling protons at the indole ring (H-4, -5, -6, and -7) appeared as two doublets and two triplets, all with coupling constants of 7–9 Hz in the 1H NMR spectra of1a,2a,and7a–11a(data not shown). In com-parison, the two triplets had disappeared in the1H NMR spec-tra of their enzyme products1b,2b, and7b–11b. One addi-tional singlet or doublet with a coupling constant smaller than 2 Hz was observed instead. These changes indicated that prenyl-ations had taken place at C-5 or C-6. As given in Table 2, the signals of the remaining three protons at H-4, H-5 or H-6 and H-7 in the1H NMR spectra (all taken in CD3OD) were found to be in the same order, i.e. (from low to high magnetic field) doublet (1.6 Hz) or broad singlet, doublet (8.3– 8.4 Hz), and double doublet (8.3– 8.4 and 1.5–1.6 Hz). The singlets for H-2 were found between the doublet (8.3– 8.4 Hz) and double dou-blet. It is plausible that the structures of these compounds have the same prenylation position.
TABLE 1
Enzyme activity of 5-DMATS towards tryptophan and other simple indole derivatives (1a–12a)
The reaction mixtures containing indole derivatives and DMAPP were incubated with 1Mprotein for 7 h. Under this condition,L-tryptophan was completely consumed.
TABLE 2
Signals for aromatic protons in the1H NMR spectra of the selected products (CD3OD)
For structures, see Fig. 3. The signals are arranged in an order from low to high magnetic field so that the relative signal positions caused by C5-prenylation can be better compared.
# ,Due to the presence of a double bond between C-10 and C-11, the signal of H-2 was downshifted in the1H NMR spectrum.
1376 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 2 •JANUARY 6, 2012
Enzyme products of1aand7awith prenylation at C-6 were reported by Takahashiet al.(13). The NMR data of 6-dimethyl-allyl-L-tryptophan and 6-dimethylallyl-L-abrine (also taken in CD3OD) differed clearly from those of1band7b, especially in the region for aromatic protons. The aromatic protons in the
1H NMR spectra of 6-dimethylallyl-L-tryptophan and 6-di-methylallyl-L-abrine appeared (from low to high magnetic field) in a different order than in the spectra of1band7b,i.e.doublet (7.8 – 8.2 Hz), two singlets, and double doublet. Therefore, the prenylation position in1band7bmust be C-5. This conclusion was also confirmed by interpretation of the two-dimensional NMR spectra of1b(in DMSO-d6) and7b(in CD3OD). In the HMBC spectrum of1b(seesupplemental Figs. S2 and S3), con-nectivity from␦H7.32 (1H, br. s, H-4) to C-1⬘of the prenyl moiety at␦C34.1 proved unequivocally the attachment of the dimethylallyl moiety to C-5. The signal at␦H7.32 (1H, s) for proton H-4 was unambiguously confirmed by the detected con-nectivities between this signal and C-8 at 134.9 ppm as well as C-3 at 109.3 ppm. For 7b, similar phenomena were also observed in the HMBC spectrum (seesupplemental Figs. S2 and S9). It can be concluded that the prenyl moieties in1b,2b, and7b–11bare attached to C-5 of the indole rings.
Substrate12ais also a derivative ofL-tryptophan with alter-ation at the side chain. Because of the presence of a double bond between C-10 and C-11, the signal of H-2 was downshifted in
1H NMR spectrum to␦H7.60 (1H, s), in comparison with those of1b,2b,and7b–11bbetween 7.00 and 7.18 ppm. However, the signals for H-4, H-6, and H-7 of12bappeared in the same order as in the spectra of1b,2b, and7b–11b(Table 2), proving the C5-prenylation in the structure of12b.
In the1H NMR spectrum of3b(seesupplemental Fig. S5), the two doublets at␦H6.85 (1H, d, 8.2 Hz, H-6) and␦H7.08 (1H, d, 8.2 Hz, H-7) represent signals for two protons at the ortho-position and indicated the prenylation at C-5 or C-7. The con-nectivity from H-6 to␦C31.4 of C-1⬘and from␦H2.55 (3H, s, H-13) of the methyl group at C-4 to ␦C 128.9 of C-5 in the HMBC spectrum confirmed the prenylation at C-5 in3b(see supplemental Fig. S2).
The structures of4b,5b, and6bwere elucidated by interpre-tation of their1H NMR spectra (seesupplemental Figs. S6 –S8).
Three singlets observed at␦H7.14 (1H, s, H-2),␦H7.44 (1H, s, H-4), and␦H7.08 (1H, s, H-7) in the1H NMR spectrum of4b proved the prenylation at C-5. In the1H NMR spectrum of5b, the two doublets at␦H7.02 (1H, d, 10.8 Hz, H-7) and␦H7.49 (1H, d, 7.3 Hz, H-4) with clearly different coupling constants are caused by the different distances of the protons to fluoro atom at C-6. In the1H NMR spectrum of the enzyme product of 6a, signals for two products6band6cwith a ratio of 1.5:1 were detected. Unfortunately, these two compounds could not be separated from each other. Based on their different contents in the mixture, we were able to identify the major product6bas C5-prenylated derivative, which is characteristic of the pres-ence of three singlets at␦H7.14 (1H, br. s), 7.14 (1H, br. s), and 6.69 (1H, br. s) for H-2, H-4, and H-6, respectively.
In conclusion, 5-DMATS catalyzed the C5-prenylation of
L-tryptophan and simple indole derivatives (Fig. 3). To the best of our knowledge, the structures1b–12bwere not described previously. The structure of6ccould not be unequivocally elu-cidated in this study. The presence of two doublets with a cou-pling constant of 8.1 Hz in the1H NMR spectrum indicated a prenylation at C-4 or C-6.
Biochemical Characterization and Kinetic Parameters of 5-DMATS—For determination of the metal ion dependence of 5-DMATS, incubations of L-tryptophan (1a) with DMAPP were carried out in the presence of different metal ions at a final concentration of 5 mM. Incubations with the chelating agent EDTA or without additives were used as controls. In the incu-bation mixture with EDTA, no decrease of the enzyme activity was observed, in comparison with that of incubation without additives. As observed for other members of the DMATS superfamily (15, 39), several divalent metal ions enhanced slightly the enzyme activity of 5-DMATS. For example, the enzyme activities with Ca2⫹and Mg2⫹were found to be 250 and 204% of that without additives, respectively.
To study the behavior of 5-DMATS toward 12 indole deriv-atives (1a–12a) and DMAPP in detail, kinetic parameters, FIGURE 3.Prenyl transfer reactions catalyzed by 5-DMATS, exemplified with simple indole derivatives 1a–12a.
JANUARY 6, 2012 •VOLUME 287 • NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 1377
including Michaelis-Menten constants (Km) and turnover numbers (kcat), were determined by Hanes-Woolf and Eadie-Hofstee plots and are given in Table 3. The reactions catalyzed by 5-DMATS apparently followed Michaelis-Menten kinetics.
TheKmvalues for DMAPP andL-tryptophan (1a)were found to be 76 and 34M, respectively, whereasKmvalues of 0.10 –1.0 mM were determined for 2a–12a, much higher than that observed for 1a. The turnover numbers were calculated for DMAPP and1aat 1.3 and 0.87 s⫺1, respectively. Lower turn-over numbers of 0.059 – 0.76 s⫺1were determined for2a–12a.
The catalytic efficiencies (kcat/Km) of 5-DMATS toward DMAPP and 1a were calculated to be 17,105 and 25,588 s⫺1䡠M⫺1, respectively. In comparison, lower catalytic efficien-cies at 155–1538 s⫺1䡠mM⫺1were found for2a–12a, only 0.6 – 6.0% that of1a.
DISCUSSION
Prenyltransferases catalyze transfer reactions of prenyl moi-eties from prenyl diphosphates to diverse aliphatic or aromatic receptors, including proteins, terpenes, benzoic acids, naphtha-lenes, flavonoids, and indole alkaloids (1, 8, 9, 40, 41). These enzymes are involved in both primary and secondary metabo-lism, play an important role in living organisms (42), and con-tribute significantly to the structural diversity of natural prod-ucts (43). Prenylated indole alkaloids represent a large group of natural products, predominantly identified as mycotoxins in Ascomycetes (1, 44). Indole prenyltransferases catalyze transfer reactions of prenyl moieties onto the indole nucleus and are involved in the biosynthesis of diverse natural products, espe-cially mycotoxins (1, 15). A large number of indole prenyltrans-ferases, mainly belonging to the DMATS superfamily from fungi of Ascomycetes, have been identified in the last years and characterized biochemically (1, 10, 11). The members of the DMATS superfamily are soluble proteins, showed usually broad substrate specificity and accepted tryptophan, simple indole derivatives, tryptophan-containing cyclic dipeptides, or other indole-containing structures as aromatic substrates and DMAPP as prenyl donor (1, 10, 11). A few examples of soluble indole prenyltransferases have also been identified in bacteria (13, 45). One of the important features of indole prenyltrans-ferases is the regioselectivity of their prenylation reactions.
With the exception for C-5, diverse indole prenyltransferases for the other six positions (N-1, C-2, C-3, C-4, C-6, and C-7) have been identified and characterized (Fig. 1). No enzyme for
prenylation at C-5 of the indole ring was reported prior to this study, although a number of C5-prenylated derivatives have been isolated from different organisms (Fig. 2). Therefore, there is a need to find enzymes for C5-prenylation at the indole nucleus, so that these enzymes could be better used as tools for chemoenzymatic synthesis of prenylated indole derivatives or even for synthesis of prenylated hydroxynaphthalenes and fla-vonoids, which have been very recently described for several members of the DMATS superfamily (46, 47).
In this study, we identified and characterized the first tryp-tophan C5-prenyltransferase 5-DMATS from A. clavatus, which catalyzes the regiospecific C5-prenylation of indole derivatives and fills herewith the last gap in the search for indole prenyltransferases regarding their prenylation positions. Blast searching in the database with 5-DMATS from A. clavatus NRRL1 revealed the presence of three orthologues in the genome sequences ofA. oryzaeRIB40 (48),A. flavusNRRL3357 (49), andA. otaeCBS 113480 (GenBankTM). Analysis of genes in the genomic region of these prenyltransferase genes in all four strains did not provide any indication for their clustering with genes for secondary metabolite biosynthesis. No C5-pre-nylated derivative was reported for these fungi. Therefore, their roles in these strains could not be predicted in this study. It seems that these genes are the results of redundant copies in the evolution. Nevertheless, considering the lowKmvalues of 34 and 76MforL-tryptophan and DMAPP, respectively, as well as the high turnover number of 1.1 s⫺1 (Table 3), it can be speculated thatL-tryptophan and DMAPP are very likely the natural substrates of 5-DMATS in the four fungal strains. Inac-tivation of these genes in the fungal strains could provide detailed information about their roles in nature, if the genes are expressed and the gene products involved in the biosynthesis of certain substance. Therefore, we tried to detect the production of C5-prenylated indole derivatives byA. clavatusNRRL1. For this purpose, the fungus was cultivated in different liquid media like YME. Culture filtrates and mycelia were extracted with ethyl acetate and methanol, respectively. In the1H NMR spec-tra of both exspec-tracts, no signal for aromatic protons was observed, which could be assigned to C5-substituted indole rings. Signals for prenyl moieties were also absent in their1H NMR spectra (data not shown). This means that C5-substituted indole derivatives were not or only in a very low yield produced by this fungus under the tested conditions. These results could indicate that 5-DMATS is not involved in the biosynthesis of secondary metabolites inA. clavatus. It cannot be excluded, however, that 5-DMATS catalyzed a C5-prenylation in the bio-synthesis of a fungal product. It is plausible that the expression level of5-dmatsand other related genes would be too low to produce a substantial amount of prenylated derivative. In both cases, cultivation ofA. clavatusNRRL1 and5-dmats-defective mutants would very likely not result in significant changes of secondary metabolite accumulation, which prohibited the potential usage of knock-out experiments to prove gene func-tion. Therefore, optimization of culture conditions should be carried out to improve the level of gene expression inA. clava-tus. Cultivation ofA. oryzae,A. flavus,orA. otaeunder different conditions and proof of the accumulated C5-prenylated sec-ondary metabolites,e.g.by NMR analysis, after purification or TABLE 3
Kinetic parameters of 5-DMATS for selected substrates
Substrate Km kcat kcat/Km
mM s⫺1 s⫺1䡠M⫺1
L-Tryptophan (1a) 0.034 0.87 25,588
1-Methyl-DL-tryptophan (2a) 0.10 0.059 590 4-Methyl-DL-tryptophan (3a) 0.25 0.29 1160 6-Methyl-DL-tryptophan (4a) 1.0 0.76 760 6-Fluoro-DL-tryptophan (5a) 0.27 0.29 1074 7-Methyl-DL-tryptophan (6a) 0.47 0.40 851
L-Abrine (7a) 0.26 0.40 1538
N-Acetyl-DL-tryptophan (8a) 0.35 0.10 286
L--Homotryptophan (9a) 0.97 0.15 155
DL-Indole-3-lactic acid (10a) 0.67 0.43 642 Indole-3-propionic acid (11a) 0.39 0.39 1000 trans-Indole-3-acrylic acid (12a) 0.40 0.14 350
DMAPP 0.076 1.3 17,105
1378 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 2 •JANUARY 6, 2012